Male reproduction is regulated by a number of essential and specific processes. The highly evolutionarily conserved piRNA pathway is essential for male fertility in mice. MOV10L1, a putative RNA helicase, is required for biogenesis of all piRNAs and thus is a master regulator of this pathway. MOV10L1 interacts with piRNA- binding proteins, including PIWIL1 and PIWIL2, and these interactions are critical for piRNA biogenesis, and thus male fertility. Meiotic recombination is responsible for exchange of genetic materials between homologous chromosomes and faithful chromosome segregation during meiosis. MEIOB, a single-stranded DNA-binding protein, is a novel meiosis-specific factor essential for meiotic recombination. MEIOB interacts with SPATA22, another meiosis-specific factor, and we find that this interaction is critical for their mutual stability. Notably, all thes proteins (MOV10L1, PIWIL1, PIWIL2, MEIOB, and SPATA22) are germ cell-specific and knockout mutant mice for any of these genes are viable but sterile due to arrest in meiosis or spermiogenesis. As these mutant mice are otherwise healthy, their infertility demonstrates a pure sterile phenotype. Based on our genetic and biochemical studies, we hypothesize that the protein components in the piRNA pathway and meiotic recombination are excellent targets for male contraception with minimal side effects. Here, we specifically propose to validate MOV10L1/Piwi and MEIOB/SPATA22 protein-protein interactions as novel male contraceptive targets and to screen for small molecule inhibitors of these interactions. We will systematically map the binding domains in MOV10L1/PIWIL1/2 (Aim 1) and in MEIOB/SPATA22 (Aim 2), and develop powerful bimolecular fluorescence complementation (BiFC) assays to monitor MOV10L1-Piwi and MEIOB-SPATA22 interactions. In Aim 3, we will perform high throughput cell-based BiFC screens of libraries of small molecule compounds for inhibitors of MOV10L1-Piwi and MEIOB-SPATA22 interactions. With genetics as the foundation, our innovative cell-based screens will identify lead compounds that inhibit these novel male contraceptive targets for future development of an orally active non-hormonal contraceptive.